Much of our present knowledge regarding the structure and function of deoxyribonucleic acid (hereinafter "DNA") has come from studying the genetic coding properties of DNA isolated from prokaryotic and eukaryotic genomes. It is known that the single chromosome of prokaryotes contains a single DNA molecule which is almost entirely a unique, a non-repeated sequence of a few hundred thousand to a few million base pairs in sequence. The chromosomes of eukaryotes, being much more complex, generally contain much more DNA per genome than is needed to code for all the proteins required for cell function. Eukaryotic DNA sequences have thus been divided into three categories based on the degree of their reiteration in the genome: single copy DNA, which includes the coding sequences for the main proteins of the cell; these single copy DNA sequences characteristically comprise 40-80% of the total DNA in the cell. Moderately repeated DNA includes those DNA sequences that are repeated from 2 to 10.sup.5 times within the genome; some of these sequences code for major macromolecules of the cell including ribosomal and transfer RNAs and such proteins as histones, globins or immunoglobulins, while other sequences may play a regulatory or structural role in the genome. Highly repeated DNA represents those DNA sequences which have been repeated in greater than 10.sup.5 copies; the function of these sequences is not clearly understood at the present time. Both moderately and highly repeated DNA sequences can be arranged in either of the two different modes. In the first type of arrangement, the basic structural units of a repeated sequence are interspersed at different locations in the genome and linked to single copy sequences. In the second type of arrangement, the basic structural units are linked to each other in long tandem arrays. When total genomic DNA is digested with a restriction enzyme, that disrupts DNA at specific sites, the interspersed repeated sequences give rise to a series of identical restriction fragments only if the basic structural unit of such sequences contains two or more sites that are recognized by the restriction enzyme; if less than two restriction sites are present within the structural unit, the restriction enzyme digestion gives rise to a heterogeneous mixture of fragments containing different single copy DNA sequences linked to the same repeated sequence. On the other hand, the presence of only one restriction site in the basic structural unit of a tandemly repeated sequence is sufficient to convert such a sequence into a series of identical restriction fragments. This differential response to restriction enzyme digestion is important in the determination of the type of arrangement of a repeated DNA sequence in the genome.
Much of our present knowledge regarding individual DNA sequences comes from experiments in which genomic DNA is digested using restriction endonucleases followed by agarose gel electrophoretic separation of the DNA fragments. The individual bands containing DNA fragments of various molecular weight have been immobilized by transfer onto nitrocellulose filters [Southern, J. Mol. Biol. 98:503-517 (1975)] or in dried agarose gels [Shinnick et al., Nuc. Acids Res. 2:1911-1929 (1975)] and the restriction fragments subsequently hybridized by a combination with exogenously added DNA or RNA probes containing a radioactive label. After washing out the nonhybridized radioactive material, the locations of the hybrids are determined by autoradiography. These techniques, however, require that the DNA or RNA sequences in the probe be complementary to the DNA in the restriction fragments only at a limited number of sites so that an interpretable pattern can be obtained upon autoradiography. There is no requirement (and often no desire) for complete homology in the probe for the DNA sequences in the restriction fragments.
The ability of the probe to hybridize with less than completely homologous DNA fragments often creates a severe problem when the probe comprises a genomic clone of eukaryotic DNA for hybridization with a restriction digest of total genomic DNA. Under these conditions, the frequent presence within the cloned probe of short interspersed repeated DNA sequences results in a smeared autoradiography pattern due to hybridization of the repeated sequences within the probe with a great number of different restriction fragments containing the same repeated sequence linked to different single copy sequences [Fisher et al., Proc. Natl. Acad. Sci. USA 81:520-524 (1984)]. The data obtained under such test conditions is often confusing and frequently undecipherable.
The deficiencies of presently known methods of analyses and detection become even more apparent in view of recent studies which demonstrated that selective amplification of specific DNA sequences is a common mechanism for adaptation of eukaryotic cells to a variety of selective conditions including drug resistance [Stark and Wahl, Ann. Rev. Biochem. 53:447-495 (1984)]. Amplification of individual genes was also found to occur in some developmental processes and has been suggested as a mechanism in carcinogenesis and tumor progression [Cowell, Ann. Rev. Genet. 16:21-59 (1982); Pall, Proc. Natl. Acad. Sci. USA 78:2465-2468 (1981); Varshavsky, Cell 25:561-572 (1981)]. The existing DNA hybridization techniques provide for the detection of amplified genes only if such genes have already been cloned and are available for use as probes. On the other hand, amplification of certain uncloned genes can provide a unique opportunity for their isolation, given the possibility of identification of amplified DNA sequences within the genome solely on the basis of their amplification. Amplified DNA sequences are defined as those sequences that have become reiterated relatively recently, e.g. in the course of selection for drug resistance or in the course of carcinogenesis, as opposed to the repeated DNA sequences that are always found in multiple copies in the genome of a given organism. The existing approaches towards this goal have included the purification of chromosomal structures known to contain amplified DNA [George and Powers, Cell 24:117-123 (1981); Kanda et al., Proc. Natl. Acad. Sci. USA 80:4069-4073 (1983)] and the cloning of amplified DNA sequences after differential screening with genomic probes [Brison et al., Mol. Cell, Biol. 2:578-587 (1982)]. None of these techniques provide for the rapid detection of amplified genes in cellular DNA preparations or comparison of amplified individual DNA sequences between different DNA preparations prior to cloning such amplified DNA sequences and their subsequent use as probes. A more general approach involves detection of highly amplified restriction fragments of DNA as bands that become detectable upon ethidium bromide staining of the gel [Heintz & Hamlin, Proc. Natl. Acad. Sci. USA 79:4083-4087 (1982); Tyler-Smith & Bostock, J. Mol. Biol. 153:203-218 (1982)]. The sensitivity of this method, however, is very low in that the DNA sequences within the restriction fragments must be repeated at least several hundred times per mammalian genome before they can be detected as distinct bands against the background produced by heterogeneous single copy DNA fragments.
It is apparent, therefore, that there is a substantial need for a general method for the detection and characterization of amplified individual nucleic acid sequences, especially for the analysis of those systems where the nature of the amplified genes is presently unknown and/or there are no cloned probes yet available. The need for a sensitive and precise assay method is most critical when the selectively amplified DNA sequences have been amplified from two to two hundred times, as is the situation in most cases of gene amplification.